Genetically modified mice that produce hybrid antibodies

ABSTRACT

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/719,842, filed Dec. 19, 2012 and U.S. patent application Ser. No.13/719,819, filed on Dec. 19, 2012, both of which are continuations ofU.S. patent application Ser. No. 13/154,976, filed Jun. 7, 2011, whichis a continuation of U.S. patent application Ser. No. 11/595,427, filedNov. 9, 2006, which is a continuation of U.S. patent application Ser.No. 10/624,044, filed Jul. 21, 2003, now abandoned, which is adivisional of U.S. patent application Ser. No. 09/784,859, filed Feb.16, 2001, now U.S. Pat. No. 6,596,541; each of which is incorporated byreference herein.

FIELD OF THE INVENTION

The field of this invention is a method for engineering and utilizinglarge DNA vectors to target, via homologous recombination, and modify,in any desirable fashion, endogenous genes and chromosomal loci ineukaryotic cells. The field also encompasses the use of these cells togenerate organisms bearing the genetic modification, the organisms,themselves, and methods of use thereof.

BACKGROUND

The use of LTVECs provides substantial advantages over current methods.For example, since these are derived from DNA fragments larger thanthose currently used to generate targeting vectors, LTVECs can be morerapidly and conveniently generated from available libraries of largegenomic DNA fragments (such as BAC and PAC libraries) than targetingvectors made using current technologies. In addition, largermodifications as well as modifications spanning larger genomic regionscan be more conveniently generated than using current technologies.Furthermore, the present invention takes advantage of long regions ofhomology to increase the targeting frequency of “hard to target” loci,and also diminishes the benefit, if any, of using isogenic DNA in thesetargeting vectors.

The present invention thus provides for a rapid, convenient, andstreamlined method for systematically modifying virtually all theendogenous genes and chromosomal loci of a given organism.

Gene targeting by means of homologous recombination between homologousexogenous DNA and endogenous chromosomal sequences has proven to be anextremely valuable way to create deletions, insertions, designmutations, correct gene mutations, introduce transgenes, or make othergenetic modifications in mice. Current methods involve using standardtargeting vectors, with regions of homology to endogenous DNA typicallytotaling less than 10-20 kb, to introduce the desired geneticmodification into mouse embryonic stem (ES) cells, followed by theinjection of the altered ES cells into mouse embryos to transmit theseengineered genetic modifications into the mouse germline (Smithies etal., Nature, 317:230-234, 1985; Thomas et al., Cell, 51:503-512, 1987;Koller et al., Proc Natl Acad Sci USA, 86:8927-8931, 1989; Kuhn et al.,Science, 254:707-710, 1991; Thomas et al., Nature, 346:847-850, 1990;Schwartzberg et al., Science, 246:799-803, 1989; Doetschman et al.,Nature, 330:576-578, 1987; Thomson et al., Cell, 5:313-321, 1989;DeChiara et al., Nature, 345:78-80, 1990; U.S. Pat. No. 5,789,215,issued Aug. 4, 1998 in the name of GenPharm International) In thesecurrent methods, detecting the rare ES cells in which the standardtargeting vectors have correctly targeted and modified the desiredendogenous gene(s) or chromosomal locus (loci) requires sequenceinformation outside of the homologous targeting sequences containedwithin the targeting vector. Assays for successful targeting involvestandard Southern blotting or long PCR (see for example Cheng, et al.,Nature, 369:684-5, 1994; U.S. Pat. No. 5,436,149) from sequences outsidethe targeting vector and spanning an entire homology arm (seeDefinitions); thus, because of size considerations that limit thesemethods, the size of the homology arms are restricted to less than 10-20kb in total (Joyner, The Practical Approach Series, 293, 1999).

The ability to utilize targeting vectors with homology arms larger thanthose used in current methods would be extremely valuable. For example,such targeting vectors could be more rapidly and conveniently generatedfrom available libraries containing large genomic inserts (e.g. BAC orPAC libraries) than targeting vectors made using current technologies,in which such genomic inserts have to be extensively characterized andtrimmed prior to use. In addition, larger modifications as well asmodifications spanning larger genomic regions could be more convenientlygenerated and in fewer steps than using current technologies.Furthermore, the use of long regions of homology could increase thetargeting frequency of “hard to target” loci in eukaryotic cells, sincethe targeting of homologous recombination in eukaryotic cells appears tobe related to the total homology contained within the targeting vector(Deng and Capecchi, Mol Cell Biol, 12:3365-71, 1992). In addition, theincreased targeting frequency obtained using long homology arms coulddiminish any potential benefit that can be derived from using isogenicDNA in these targeting vectors.

The problem of engineering precise modifications into very large genomicfragments, such as those cloned in BAC libraries, has largely beensolved through the use of homologous recombination in bacteria (Zhang,et al., Nat Genet, 20:123-8, 1998; Yang, et al., Nat Biotechnol,15:859-65, 1997; Angrand, et al., Nucleic Acids Res, 27:e16, 1999;Muyrers, et al., Nucleic Acids Res, 27:1555-7, 1999; Narayanan, et al.,Gene Ther, 6:442-7, 1999), allowing for the construction of vectorscontaining large regions of homology to eukaryotic endogenous genes orchromosomal loci. However, once made, these vectors have not beengenerally useful for modifying endogenous genes or chromosomal loci viahomologous recombination because of the difficulty in detecting rarecorrect targeting events when homology arms are larger than 10-20 kb(Joyner supra). Consequently, vectors generated using bacterialhomologous recombination from BAC genomic fragments must still beextensively trimmed prior to use as targeting vectors (Hill et al.,Genomics, 64:111-3, 2000). Therefore, there is still a need for a rapidand convenient methodology that makes possible the use of targetingvectors containing large regions of homology so as to modify endogenousgenes or chromosomal loci in eukaryotic cells.

In accordance with the present invention, Applicants provide novelmethods that enables the use of targeting vectors containing largeregions of homology so as to modify endogenous genes or chromosomal lociin eukaryotic cells via homologous recombination. Such methods overcomethe above-described limitations of current technologies. In addition,the skilled artisan will readily recognize that the methods of theinvention are easily adapted for use with any genomic DNA of anyeukaryotic organism including, but not limited to, animals such asmouse, rat, other rodent, or human, as well as plants such as soy, cornand wheat.

SUMMARY OF THE INVENTION

In accordance with the present invention, Applicants have developed anovel, rapid, streamlined, and efficient method for creating andscreening eukaryotic cells which contain modified endogenous genes orchromosomal loci. This novel methods combine, for the first time: 1.Bacterial homologous recombination to precisely engineer a desiredgenetic modification within a large cloned genomic fragment, therebycreating a large targeting vector for use in eukaryotic cells (LTVECs);2. Direct introduction of these LTVECs into eukaryotic cells to modifythe endogenous chromosomal locus of interest in these cells; and 3. Ananalysis to determine the rare eukaryotic cells in which the targetedallele has been modified as desired, involving an assay for modificationof allele (MOA) of the parental allele that does not require sequenceinformation outside of the targeting sequence, such as, for example,quantitative PCR.

A preferred embodiment of the invention is a method for geneticallymodifying an endogenous gene or chromosomal locus in eukaryotic cells,comprising: a) obtaining a large cloned genomic fragment containing aDNA sequence of interest; b) using bacterial homologous recombination togenetically modify the large cloned genomic fragment of (a) to create alarge targeting vector for use in the eukaryotic cells (LTVEC); c)introducing the LTVEC of (b) into the eukaryotic cells to modify theendogenous gene or chromosomal locus in the cells; and d) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (c) to identify those eukaryotic cells in which theendogenous gene or chromosomal locus has been genetically modified.Another embodiment of the invention is a method wherein the geneticmodification to the endogenous gene or chromosomal locus comprisesdeletion of a coding sequence, gene segment, or regulatory element;alteration of a coding sequence, gene segment, or regulatory element;insertion of a new coding sequence, gene segment, or regulatory element;creation of a conditional allele; or replacement of a coding sequence orgene segment from one species with an homologous or orthologous codingsequence from a different species. An alternative embodiment of theinvention is a method wherein the alteration of a coding sequence, genesegment, or regulatory element comprises a substitution, addition, orfusion, wherein the fusion comprises an epitope tag or bifunctionalprotein. Yet another embodiment of the invention is a method wherein thequantitative assay comprises quantitative PCR, comparative genomichybridization, isothermic DNA amplification, or quantitativehybridization to an immobilized probe, wherein the quantitative PCRcomprises TAQMAN® technology or quantitative PCR using molecularbeacons. Another preferred embodiment of the invention is a methodwherein the eukaryotic cell is a mammalian embryonic stem cell and inparticular wherein the embryonic stem cell is a mouse, rat, or otherrodent embryonic stem cell. Another preferred embodiment of theinvention is a method wherein the endogenous gene or chromosomal locusis a mammalian gene or chromosomal locus, preferably a human gene orchromosomal locus or a mouse, rat, or other rodent gene or chromosomallocus. An additional preferred embodiment is one in which the LTVEC iscapable of accommodating large DNA fragments greater than 20 kb, and inparticular large DNA fragments greater than 100 kb. Another preferredembodiment is a genetically modified endogenous gene or chromosomallocus that is produced by the method of the invention. Yet anotherpreferred embodiment is a genetically modified eukaryotic cell that isproduced by the method of the invention. A preferred embodiment of theinvention is a non-human organism containing the genetically modifiedendogenous gene or chromosomal locus produced by the method of theinvention. Also preferred in a non-human organism produced from thegenetically modified eukaryotic cells or embryonic stem cells producedby the method of the invention.

A preferred embodiment is a non-human organism containing a geneticallymodified endogenous gene or chromosomal locus, produced by a methodcomprising the steps of: a) obtaining a large cloned genomic fragmentcontaining a DNA sequence of interest; b) using bacterial homologousrecombination to genetically modify the large cloned genomic fragment of(a) to create a large targeting vector (LTVEC) for use in embryonic stemcells; c) introducing the LTVEC of (b) into the embryonic stem cells tomodify the endogenous gene or chromosomal locus in the cells; d) using aquantitative assay to detect modification of allele (MOA) in theembryonic stem cells of (c) to identify those embryonic stem cells inwhich the endogenous gene or chromosomal locus has been geneticallymodified; e) introducing the embryonic stem cell of (d) into ablastocyst; and f) introducing the blastocyst of (e) into a surrogatemother for gestation.

An additional preferred embodiment of the invention is a non-humanorganism containing a genetically modified endogenous gene orchromosomal locus, produced by a method comprising the steps of: a)obtaining a large cloned genomic fragment containing a DNA sequence ofinterest; b) using bacterial homologous recombination to geneticallymodify the large cloned genomic fragment of (a) to create a largetargeting vector for use in eukaryotic cells (LTVEC); c) introducing theLTVEC of (b) into the eukaryotic cells to genetically modify theendogenous gene or chromosomal locus in the cells; d) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (c) to identify those eukaryotic cells in which theendogenous gene or chromosomal locus has been genetically modified; e)removing the nucleus from the eukaryotic cell of (d); f) introducing thenucleus of (e) into an oocyte; and g) introducing the oocyte of (f) intoa surrogate mother for gestation.

Yet another preferred embodiment is a non-human organism containing agenetically modified endogenous gene or chromosomal locus, produced by amethod comprising the steps of: a) obtaining a large cloned genomicfragment containing a DNA sequence of interest; b) using bacterialhomologous recombination to genetically modify the large cloned genomicfragment of (a) to create a large targeting vector for use in eukaryoticcells (LTVEC); c) introducing the LTVEC of (b) into the eukaryotic cellsto genetically modify the endogenous gene or chromosomal locus in thecells; d) using a quantitative assay to detect modification of allele(MOA) in the eukaryotic cells of (c) to identify those eukaryotic cellsin which the endogenous gene or chromosomal locus has been geneticallymodified; e) fusing the eukaryotic cell of (d) with another eukaryoticcell; f) introducing the fused eukaryotic cell of (e) into a surrogatemother for gestation.

A preferred embodiment of the invention is a method for geneticallymodifying an endogenous gene or chromosomal locus of interest in mouseembryonic stem cells, comprising: a) obtaining a large cloned genomicfragment greater than 20 kb which contains a DNA sequence of interest,wherein the large cloned DNA fragment is homologous to the endogenousgene or chromosomal locus; b) using bacterial homologous recombinationto genetically modify the large cloned genomic fragment of (a) to createa large targeting vector for use in the mouse embryonic stem cells,wherein the genetic modification is deletion of a coding sequence, genesegment, or regulatory element; c) introducing the large targetingvector of (b) into the mouse embryonic stem cells to modify theendogenous gene or chromosomal locus in the cells; and d) using aquantitative assay to detect modification of allele (MOA) in the mouseembryonic stem cells of (c) to identify those mouse embryonic stem cellsin which the endogenous gene or chromosomal locus has been geneticallymodified, wherein the quantitative assay is quantitative PCR. Alsopreferred is a genetically modified mouse embryonic stem cell producedby this method; a mouse containing a genetically modified endogenousgene or chromosomal locus produced by this method; and a mouse producedfrom the genetically modified mouse embryonic stem cell.

Another preferred embodiment is a mouse containing a geneticallymodified endogenous gene or chromosomal locus of interest, produced by amethod comprising the steps of: a) obtaining a large cloned genomicfragment greater than 20 kb which contains a DNA sequence of interest,wherein the large cloned DNA fragment is homologous to the endogenousgene or chromosomal locus; b) using bacterial homologous recombinationto genetically modify the large cloned genomic fragment of (a) to createa large targeting vector for use in the mouse embryonic stem cells,wherein the genetic modification is deletion of a coding sequence, genesegment, or regulatory element; c) introducing the large targetingvector of (b) into the mouse embryonic stem cells to modify theendogenous gene or chromosomal locus in the cells; and d) using aquantitative assay to detect modification of allele (MOA) in the mouseembryonic stem cells of (c) to identify those mouse embryonic stem cellsin which the endogenous gene or chromosomal locus has been geneticallymodified, wherein the quantitative assay is quantitative PCR; e)introducing the mouse embryonic stem cell of (d) into a blastocyst; andf) introducing the blastocyst of (e) into a surrogate mother forgestation.

One embodiment of the invention is a method of replacing, in whole or inpart, in a non-human eukaryotic cell, an endogenous immunoglobulinvariable region gene locus with an homologous or orthologous human genelocus comprising: a) obtaining a large cloned genomic fragmentcontaining, in whole or in part, the homologous or orthologous humangene locus; b) using bacterial homologous recombination to geneticallymodify the cloned genomic fragment of (a) to create a large targetingvector for use in the eukaryotic cells (LTVEC); c) introducing the LTVECof (b) into the eukaryotic cells to replace, in whole or in part, theendogenous immunoglobulin variable gene locus; and d) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (c) to identify those eukaryotic cells in which theendogenous immunoglobulin variable region gene locus has been replaced,in whole or in part, with the homologous or orthologous human genelocus.

Another embodiment is a method of replacing, in whole or in part, in anon-human eukaryotic cell, an endogenous immunoglobulin variable regiongene locus with an homologous or orthologous human gene locus furthercomprising the steps: e) obtaining a large cloned genomic fragmentcontaining a part of the homologous or orthologous human gene locus thatdiffers from the fragment of (a); f) using bacterial homologousrecombination to genetically modify the cloned genomic fragment of (e)to create a second LTVEC; g) introducing the second LTVEC of (f) intothe eukaryotic cells identified in step (d) to replace, in whole or inpart, the endogenous immunoglobulin variable gene locus; and h) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (g) to identify those eukaryotic cells in which theendogenous immunoglobulin variable region gene locus has been replaced,in whole or in part, with the homologous or orthologous human genelocus.

Another embodiment of the above method is a method wherein steps (e)through (h) are repeated until the endogenous immunoglobulin variableregion gene locus is replaced in whole with an homologous or orthologoushuman gene locus.

Another embodiment of the method is one in which the immunoglobulinvariable gene locus is a locus selected from the group consisting of a)a variable gene locus of the kappa light chain; b) a variable gene locusof the lambda light chain; and c) a variable gene locus of the heavychain.

A preferred embodiment is a method wherein the quantitative assaycomprises quantitative PCR, FISH, comparative genomic hybridization,isothermic DNA amplification, or quantitative hybridization to animmobilized probe, and in particular wherein the quantitative PCRcomprises TAQMAN®. technology or quantitative PCR using molecularbeacons.

Yet another preferred embodiment is a method of replacing, in whole orin part, in a mouse embryonic stem cell, an endogenous immunoglobulinvariable region gene locus with its homologous or orthologous human genelocus comprising: a) obtaining a large cloned genomic fragmentcontaining, in whole or in part, the homologous or orthologous humangene locus; b) using bacterial homologous recombination to geneticallymodify the large cloned genomic fragment of (a) to create a largetargeting vector for use in the embryonic stem cells; c) introducing thelarge targeting vector of (b) into mouse embryonic stem cells toreplace, in whole or in part, the endogenous immunoglobulin variablegene locus in the cells; and d) using a quantitative PCR assay to detectmodification of allele (MOA) in the mouse embryonic stem cells of (d) toidentify those mouse embryonic stem cells in which the endogenousvariable gene locus has been replaced, in whole or in part, with thehomologous or orthologous human gene locus.

In another embodiment, the method further comprises: e) obtaining alarge cloned genomic fragment containing a part of the homologous ororthologous human gene locus that differs from the fragment of (a); f)using bacterial homologous recombination to genetically modify thecloned genomic fragment of (e) to create a large targeting vector foruse in the embryonic stem cells; g) introducing the large targetingvector of (f) into the mouse embryonic stem cells identified in step (d)to replace, in whole or in part, the endogenous immunoglobulin variablegene locus; and h) using a quantitative assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (g) to identify thosemouse embryonic stem cells in which the endogenous immunoglobulinvariable region gene locus has been replaced, in whole or in part, withthe homologous or orthologous human gene locus.

Another preferred embodiment is a genetically modified immunoglobulinvariable region gene locus produced by the methods described above; agenetically modified eukaryotic cell comprising a genetically modifiedimmunoglobulin variable region gene locus produced by the methodsdescribed above; a non-human organism comprising a genetically modifiedimmunoglobulin variable region gene locus produced by the methodsdescribed above; and a mouse embryonic stem cell containing agenetically modified immunoglobulin variable region gene locus producedby the methods described above.

Also preferred is an embryonic stem cell wherein the mouse heavy chainvariable region locus is replaced, in whole or in part, with a humanheavy chain variable gene locus; an embryonic stem cell of claim whereinthe mouse kappa light chain variable region locus is replaced, in wholeor in part, with a human kappa light chain variable region locus; anembryonic stem cell wherein the mouse lambda light chain variable regionlocus is replaced, in whole or in part, with a human lambda light chainvariable region locus; and an embryonic stem cell wherein the heavy andlight chain variable region gene loci are replaced, in whole, with theirhuman homologs or orthologs.

Yet another preferred embodiment is an antibody comprising a humanvariable region encoded by the genetically modified variable gene locusof described above; an antibody further comprising a non-human constantregion; and an antibody further comprising a human constant region.

Also preferred is a transgenic mouse having a genome comprising entirelyhuman heavy and light chain variable region loci operably linked toentirely endogenous mouse constant region loci such that the mouseproduces a serum containing an antibody comprising a human variableregion and a mouse constant region in response to antigenic stimulation;a transgenic mouse having a genome comprising human heavy and/or lightchain variable region loci operably linked to endogenous mouse constantregion loci such that the mouse produces a serum containing an antibodycomprising a human variable region and a mouse constant region inresponse to antigenic stimulation; a transgenic mouse containing anendogenous variable region locus that has been replaced with anhomologous or orthologous human variable locus, such mouse beingproduced by a method comprising: a) obtaining one or more large clonedgenomic fragments containing the entire homologous or orthologous humanvariable region locus; b) using bacterial homologous recombination togenetically modify the cloned genomic fragment(s) of (a) to create largetargeting vector(s) for use in mouse embryonic stem cells; c)introducing the large targeting vector(s) of (b) into mouse embryonicstem cells to replace the entire endogenous variable region locus in thecells; and d) using a quantitative PCR assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (c) to identify thosemouse embryonic stem cells in which the entire endogenous variableregion locus has been replaced with the homologous or orthologous humanvariable region locus; e) introducing the mouse embryonic stem cell of(d) into a blastocyst; and f) introducing the blastocyst of (e) into asurrogate mother for gestation.

Still yet another preferred embodiment of the invention is a method ofmaking a human antibody comprising: a) exposing the mouse describedabove to antigenic stimulation, such that the mouse produces an antibodyagainst the antigen; b) isolating the DNA encoding the variable regionsof the heavy and light chains of the antibody; c) operably linking theDNA encoding the variable regions of (b) to DNA encoding the human heavyand light chain constant regions in a cell capable of expressing activeantibodies; d) growing the cell under such conditions as to express thehuman antibody; and e) recovering the antibody. In another preferredembodiment, the cell described above is a CHO cell. Also preferred is amethod of wherein the DNA of step (b) described above is isolated from ahybridoma created from the spleen of the mouse exposed to antigenicstimulation in step (a) described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic diagram of the generation of a typical LTVEC usingbacterial homologous recombination. (hb1=homology box 1; hb2=homologybox 2; RE=restriction enzyme site).

FIG. 2: Schematic diagram of donor fragment and LTVEC for mouse OCR10.(hb1=homology box 1; lacZ=β-galactosidase ORF; SV40 polyA=a DNA fragmentderived from Simian Virus 40, containing a polyadenylation site andsignal; PGKp=mouse phosphoglycerate kinase (PGK) promoter; EM7=abacterial promoter; neo=neomycin phosphotransferase; PGK polyA=3′untranslated region derived from the PGK gene and containing apolyadenylation site and signal; hb2=homology box 2)

FIG. 3A-3D: Sequence of the mouse OCR10 cDNA (upper strand, SEQ ID NO:5;amino acid, SEQ ID NO:6), homology box 1 (hb1), homology box 2 (hb2),and TAQMAN® probes and primers used in a quantitative PCR assay todetect modification of allele (MOA) in ES cells targeted using themOCR10 LTVEC. hb1: base pairs 1 to 211; hb2: base pairs 1586 to 1801;TAQMAN® probe and corresponding PCR primer set derived from mOCR10 exon3: TAQMAN® probe: nucleotides 413 to 439—upper strand; Primer ex3-5′:nucleotides 390 to 410—upper strand; Primer ex3-3′: nucleotides 445 to461—lower strand; TAQMAN® probe and corresponding PCR primer set derivedfrom mOCR10 exon 4: TAQMAN® probe: nucleotides 608 to 639—upper strand;Primer ex4-5′: nucleotides 586 to 605—upper strand; Primer ex4-3′:nucleotides 642 to 662—lower strand.

FIG. 4A-4D: (SEQ ID NO:5-6) Schematic diagram of the two LTVECsconstructed to replace the mouse VDJ region with human VDJ region. A:Large insert (BAC) clones spanning the entire VDJ region of the humanheavy chain locus are isolated. B: In this example, large insert (BAC)clones are isolated from the ends of the mouse VDJ region as a source ofhomology arms which are used to direct integration via homologousrecombination of the human VDJ sequences in a two step process. C-D: Inthe first step, LTVEC1 is constructed by bacterial homologousrecombination in E. coli. LTVEC1 contains, in order: a large mousehomology arm derived from the region upstream from the mouse DJ region,but whose absolute endpoints are not important; a cassette encoding aselectable marker functional in ES cells (PGK-neomycinR); a loxP site; alarge human insert spanning from several V gene segments through theentire DJ region; and a mouse homology arm containing the regionimmediately adjacent to, but not including, the mouse J segments. In thesecond step, LTVEC2 is constructed by bacterial homologous recombinationin E. coli. LTVEC2 contains, in order: a large mouse homology armcontaining the region adjacent to the most distal mouse V gene segment,but not containing any mouse V gene segments; a large insert containinga large number of distal human V gene segments; a mutant loxP sitecalled lox511 in the orientation opposite to that of the wild type loxPsites in LTVEC2 and LTVEC1 (this site will not recombine with wild typeloxP sites but will readily recombine with other lox511 sites); a wildtype loxP site; a second selectable marker (PGK-hygromycinR); and amouse homology arm derived from the V region, but whose absoluteendpoints are not important.

DETAILED DESCRIPTION

A “targeting vector” is a DNA construct that contains sequences“homologous” to endogenous chromosomal nucleic acid sequences flanking adesired genetic modification(s). The flanking homology sequences,referred to as “homology arms”, direct the targeting vector to aspecific chromosomal location within the genome by virtue of thehomology that exists between the homology arms and the correspondingendogenous sequence and introduce the desired genetic modification by aprocess referred to as “homologous recombination”.

“Homologous” means two or more nucleic acid sequences that are eitheridentical or similar enough that they are able to hybridize to eachother or undergo intermolecular exchange.

“Gene targeting” is the modification of an endogenous chromosomal locusby the insertion into, deletion of, or replacement of the endogenoussequence via homologous recombination using a targeting vector.

A “gene knockout” is a genetic modification resulting from thedisruption of the genetic information encoded in a chromosomal locus. A“gene knockin” is a genetic modification resulting from the replacementof the genetic information encoded in a chromosomal locus with adifferent DNA sequence. A “knockout organism” is an organism in which asignificant proportion of the organism's cells harbor a gene knockout. A“knockin organism” is an organism in which a significant proportion ofthe organism's cells harbor a gene knockin.

A “marker” or a “selectable marker” is a selection marker that allowsfor the isolation of rare transfected cells expressing the marker fromthe majority of treated cells in the population. Such marker's gene'sinclude, but are not limited to, neomycin phosphotransferase andhygromycin B phosphotransferase, or fluorescing proteins such as GFP.

An “ES cell” is an embryonic stem cell. This cell is usually derivedfrom the inner cell mass of a blastocyst-stage embryo. An “ES cellclone” is a subpopulation of cells derived from a single cell of the EScell population following introduction of DNA and subsequent selection.

A “flanking DNA” is a segment of DNA that is collinear with and adjacentto a particular point of reference.

“LTVECs” are large targeting vectors for eukaryotic cells that arederived from fragments of cloned genomic DNA larger than those typicallyused by other approaches intended to perform homologous targeting ineukaryotic cells.

“Modification of allele” (MOA) refers to the modification of the exactDNA sequence of one allele of a gene(s) or chromosomal locus (loci) in agenome. This modification of allele (MOA) includes, but is not limitedto, deletions, substitutions, or insertions of as little as a singlenucleotide or deletions of many kilobases spanning a gene(s) orchromosomal locus (loci) of interest, as well as any and all possiblemodifications between these two extremes.

“Orthologous” sequence refers to a sequence from one species that is thefunctional equivalent of that sequence in another species.

General Description

Applicants have developed a novel, rapid, streamlined, and efficientmethod for creating and screening eukaryotic cells which containmodified endogenous genes or chromosomal loci. In these cells, themodification may be gene(s) knockouts, knockins, point mutations, orlarge genomic insertions or deletions or other modifications. Thesecells may be embryonic stem cells which are useful for creating knockoutor knockin organisms and in particular, knockout or knockin mice, forthe purpose of determining the function of the gene(s) that have beenaltered, deleted and/or inserted.

The novel methods described herein combine, for the first time: 1.Bacterial homologous recombination to precisely engineer a desiredgenetic modification within a large cloned genomic DNA fragment, therebycreating a large targeting vector for use in eukaryotic cells (LTVECs);2. Direct introduction of these LTVECs into eukaryotic cells to modifythe corresponding endogenous gene(s) or chromosomal locus (loci) ofinterest in these cells; and 3. An analysis to determine the rareeukaryotic cells in which the targeted allele has been modified asdesired, involving a quantitative assay for modification of allele (MOA)of the parental allele.

It should be emphasized that previous methods to detect successfulhomologous recombination in eukaryotic cells cannot be utilized inconjunction with the LTVECs of Applicants' invention because of the longhomology arms present in the LTVECs. Utilizing a LTVEC to deliberatelymodify endogenous genes or chromosomal loci in eukaryotic cells viahomologous recombination is made possible by the novel application of anassay to determine the rare eukaryotic cells in which the targetedallele has been modified as desired, such assay involving a quantitativeassay for modification of allele (MOA) of a parental allele, byemploying, for example, quantitative PCR or other suitable quantitativeassays for MOA.

The ability to utilize targeting vectors with homology arms larger thanthose used in current methods is extremely valuable for the followingreasons: 1. Targeting vectors are more rapidly and convenientlygenerated from available libraries containing large genomic inserts(e.g. BAC or PAC libraries) than targeting vectors made using previoustechnologies, in which the genomic inserts have to be extensivelycharacterized and “trimmed” prior to use (explained in detail below). Inaddition, minimal sequence information needs to be known about the locusof interest, i.e. it is only necessary to know the approximately 80-100nucleotides that are required to generate the homology boxes (describedin detail below) and to generate probes that can be used in quantitativeassays for MOA (described in detail below). 2. Larger modifications aswell as modifications spanning larger genomic regions are moreconveniently generated and in fewer steps than using previoustechnologies. For example, the method of the invention makes possiblethe precise modification of large loci that cannot be accommodated bytraditional plasmid-based targeting vectors because of their sizelimitations. It also makes possible the modification of any given locusat multiple points (e.g. the introduction of specific mutations atdifferent exons of a multi-exon gene) in one step, alleviating the needto engineer multiple targeting vectors and to perform multiple rounds oftargeting and screening for homologous recombination in ES cells. 3. Theuse of long regions of homology (long homology arms) increase thetargeting frequency of “hard to target” loci in eukaryotic cells,consistent with previous findings that targeting of homologousrecombination in eukaryotic cells appears to be related to the totalhomology contained within the targeting vector. 4. The increasedtargeting frequency obtained using long homology arms apparentlydiminishes the benefit, if any, from using isogenic DNA in thesetargeting vectors. 5. The application of quantitative MOA assays forscreening eukaryotic cells for homologous recombination not onlyempowers the use of LTVECs as targeting vectors (advantages outlinedabove) but also reduces the time for identifying correctly modifiedeukaryotic cells from the typical several days to a few hours. Inaddition, the application of quantitative MOA does not require the useof probes located outside the endogenous gene(s) or chromosomal locus(loci) that is being modified, thus obviating the need to know thesequence flanking the modified gene(s) or locus (loci). This is asignificant improvement in the way the screening has been performed inthe past and makes it a much less labor-intensive and much morecost-effective approach to screening for homologous recombination eventsin eukaryotic cells.

Methods

Many of the techniques used to construct DNA vectors described hereinare standard molecular biology techniques well known to the skilledartisan (see e.g., Sambrook, J., E. F. Fritsch And T. Maniatis.Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and3, 1989; Current Protocols in Molecular Biology, Eds. Ausubel et al.,Greene Publ. Assoc., Wiley Interscience, NY). All DNA sequencing is doneby standard techniques using an ABI 373A DNA sequencer and Taq DideoxyTerminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City,Calif.).

Step 1. Obtain a Large Genomic DNA Clone Containing the Gene(s) orChromosomal Locus (Loci) of Interest. A gene(s) or locus (loci) ofinterest can be selected based on specific criteria, such as detailedstructural or functional data, or it can be selected in the absence ofsuch detailed information as potential genes or gene fragments becomepredicted through the efforts of the various genome sequencing projects.Importantly, it should be noted that it is not necessary to know thecomplete sequence and gene structure of a gene(s) of interest to applythe method of the subject invention to produce LTVECs. In fact, the onlysequence information that is required is approximately 80-100nucleotides so as to obtain the genomic clone of interest as well as togenerate the homology boxes used in making the LTVEC (described indetail below) and to make probes for use in quantitative MOA assays.

Once a gene(s) or locus (loci) of interest has been selected, a largegenomic clone(s) containing this gene(s) or locus (loci) is obtained.This clone(s) can be obtained in any one of several ways including, butnot limited to, screening suitable DNA libraries (e.g. BAC, PAC, YAC, orcosmid) by standard hybridization or PCR techniques, or by any othermethods familiar to the skilled artisan.

Step 2. Append Homology Boxes 1 and 2 to a Modification Cassette andGeneration of LTVEC. Homology boxes mark the sites of bacterialhomologous recombination that are used to generate LTVECs from largecloned genomic fragments (FIG. 1). Homology boxes are short segments ofDNA, generally double-stranded and at least 40 nucleotides in length,that are homologous to regions within the large cloned genomic fragmentflanking the “region to be modified”. The homology boxes are appended tothe modification cassette, so that following homologous recombination inbacteria, the modification cassette replaces the region to be modified(FIG. 1). The technique of creating a targeting vector using bacterialhomologous recombination can be performed in a variety of systems (Yanget al. supra; Muyrers et al. supra; Angrand et al. supra; Narayanan etal. supra; Yu, et al., Proc Natl Acad Sci USA, 97:5978-83, 2000). Oneexample of a favored technology currently in use is ET cloning andvariations of this technology (Yu et al. supra). ET refers to the recE(Hall and Kolodner, Proc Natl Acad Sci USA, 91:3205-9, 1994) and recTproteins (Kusano et al., Gene, 138:17-25, 1994) that carry out thehomologous recombination reaction. RecE is an exonuclease that trims onestrand of linear double-stranded DNA (essentially the donor DNA fragmentdescribed infra) 5′ to 3′, thus leaving behind a linear double-strandedfragment with a 3′ single-stranded overhang. This single-strandedoverhang is coated by recT protein, which has single-stranded DNA(ssDNA) binding activity (Kovall and Matthews, Science, 277:1824-7,1997). ET cloning is performed using E. coli that transiently expressthe E. coli gene products of recE and recT (Hall and Kolodner, Proc NatlAcad Sci USA, 91:3205-9, 1994; Clark et al., Cold Spring Harb Symp QuantBiol, 49:453-62, 1984; Noirot and Kolodner, J Biol Chem, 273:12274-80,1998; Thresher et al., J Mol Biol, 254:364-71, 1995; Kolodner et al.,Mol Microbiol, 11:23-30, 1994; Hall et al., J Bacteriol, 175:277-87,1993) and the bacteriophage lambda (λ) protein λgam (Murphy, JBacteriol, 173:5808-21, 1991; Poteete et al., J Bacteriol, 170:2012-21,1988). The kgam protein is required for protecting the donor DNAfragment from degradation by the recBC exonuclease system (Myers andStahl, Annu Rev Genet, 28:49-70, 1994) and it is required for efficientET-cloning in recBC⁺ hosts such as the frequently used E. coli strainDH10b.

The region to be modified and replaced using bacterial homologousrecombination can range from zero nucleotides in length (creating aninsertion into the original locus) to many tens of kilobases (creating adeletion and/or a replacement of the original locus). Depending on themodification cassette, the modification can result in the following: (a)deletion of coding sequences, gene segments, or regulatory elements; (b)alteration(s) of coding sequence, gene segments, or regulatory elementsincluding substitutions, additions, and fusions (e.g. epitope tags orcreation of bifunctional proteins such as those with GFP); (c) insertionof new coding regions, gene segments, or regulatory elements, such asthose for selectable marker genes or reporter genes or putting new genesunder endogenous transcriptional control; (d) creation of conditionalalleles, e.g. by introduction of loxP sites flanking the region to beexcised by Cre recombinase (Abremski and Hoess, J Biol Chem,259:1509-14, 1984), or FRT sites flanking the region to be excised byFlp recombinase (Andrews et al., Cell, 40:795-803, 1985; Meyer-Leon etal., Cold Spring Harb Symp Quant Biol, 49:797-804, 1984; Cox, Proc NatlAcad Sci USA, 80:4223-7, 1983); or (e) replacement of coding sequencesor gene segments from one species with orthologous coding sequences froma different species, e.g. replacing a murine genetic locus with theorthologous human genetic locus to engineer a mouse where thatparticular locus has been ‘humanized’.

Any or all of these modifications can be incorporated into a LTVEC. Aspecific example in which an endogenous coding sequence is entirelydeleted and simultaneously replaced with both a reporter gene as well asa selectable marker is provided below in Example 1, as are theadvantages of the method of the invention as compared to previoustechnologies.

Step 3 (Optional). Verify that Each LTVEC has been Engineered Correctly.Verify that each LTVEC has been engineered correctly by: a. DiagnosticPCR to verify the novel junctions created by the introduction of thedonor fragment into the gene(s) or chromosomal locus (loci) of interest.The PCR fragments thus obtained can be sequenced to further verify thenovel junctions created by the introduction of the donor fragment intothe gene(s) or chromosomal locus (loci) of interest. b. Diagnosticrestriction enzyme digestion to make sure that only the desiredmodifications have been introduced into the LTVEC during the bacterialhomologous recombination process. c. Direct sequencing of the LTVEC,particularly the regions spanning the site of the modification to verifythe novel junctions created by the introduction of the donor fragmentinto the gene(s) or chromosomal locus (loci) of interest.

Step 4. Purification, Preparation, and Linearization of LTVEC DNA forIntroduction into Eukaryotic Cells. a. Preparation of LTVEC DNA: Prepareminiprep DNA (Sambrook et al. supra; Tillett and Neilan, Biotechniques,24:568-70, 572, 1998; of the selected LTVEC and re-transform theminiprep LTVEC DNA into E. coli using electroporation (Sambrook et al.supra). This step is necessary to get rid of the plasmid encoding therecombinogenic proteins that are utilized for the bacterial homologousrecombination step. It is useful to get rid of this plasmid (a) becauseit is a high copy number plasmid and may reduce the yields obtained inthe large scale LTVEC preps; (b) to eliminate the possibility ofinducing expression of the recombinogenic proteins; and (c) because itmay obscure physical mapping of the LTVEC. Before introducing the LTVECinto eukaryotic cells, larger amounts of LTVEC DNA are prepared bystandard methodology; Sambrook et al. supra; Tillett and Neilan,Biotechniques, 24:568-70, 572, 1998). However, this step can be bypassedif a bacterial homologous recombination method that utilizes arecombinogenic prophage is used, i.e. where the genes encoding therecombinogenic proteins are integrated into the bacterial chromosome(Yu, et al. supra), is used.

b. Linearizing the LTVEC DNA: To prepare the LTVEC for introduction intoeukaryotic cells, the LTVEC is preferably linearized in a manner thatleaves the modified endogenous gene(s) or chromosomal locus (loci) DNAflanked with long homology arms. This can be accomplished by linearizingthe LTVEC, preferably in the vector backbone, with any suitablerestriction enzyme that digests only rarely. Examples of suitablerestriction enzymes include NotI, PacI, SfiI, SrfI, SwaI, FseI, etc. Thechoice of restriction enzyme may be determined experimentally (i.e. bytesting several different candidate rare cutters) or, if the sequence ofthe LTVEC is known, by analyzing the sequence and choosing a suitablerestriction enzyme based on the analysis. In situations where the LTVEChas a vector backbone containing rare sites such as CosN sites, then itcan be cleaved with enzymes recognizing such sites, for example λterminase (Shizuya et al., Proc Natl Acad Sci USA, 89:8794-7, 1992;Becker and Gold, Proc Natl Acad Sci USA, 75:4199-203, 1978; Rackwitz etal., Gene, 40:259-66, 1985).

Step 5. Introduction of LTVEC into Eukaryotic Cells and Selection ofCells where Successful Introduction of the LTVEC has Taken Place. LTVECDNA can be introduced into eukaryotic cells using standard methodology,such as transfection mediated by calcium phosphate, lipids, orelectroporation (Sambrook et al. supra). The cells where the LTVEC hasbeen introduced successfully can be selected by exposure to selectionagents, depending on the selectable marker gene that has been engineeredinto the LTVEC. For example, if the selectable marker is the neomycinphosphotransferase (neo) gene (Beck, et al., Gene, 19:327-36, 1982),then cells that have taken up the LTVEC can be selected inG418-containing media; cells that do not have the LTVEC will die whereascells that have taken up the LTVEC will survive (Santerre, et al., Gene,30:147-56, 1984). Other suitable selectable markers include any drugthat has activity in eukaryotic cells, such as hygromycin B (Santerre,et al., Gene, 30:147-56, 1984; Bernard, et al., Exp Cell Res,158:237-43, 1985; Giordano and McAllister, Gene, 88:285-8, 1990),Blasticidin S (Izumi, et al., Exp Cell Res, 197:229-33, 1991), and otherwhich are familiar to those skilled in the art.

Step 6. Screen for Homologous Recombination Events in Eukaryotic CellsUsing Quantitative Assay for Modification of Allele (MOA). Eukaryoticcells that have been successfully modified by targeting the LTVEC intothe locus of interest can be identified using a variety of approachesthat can detect modification of allele within the locus of interest andthat do not depend on assays spanning the entire homology arm or arms.Such approaches can include but are not limited to: (a) quantitative PCRusing TAQMAN® (Lie and Petropoulos, Curr Opin Biotechnol, 9:43-8, 1998);(b) quantitative MOA assay using molecular beacons (Tan, et al.,Chemistry, 6:1107-11, 2000); (c) fluorescence in situ hybridization FISH(Laan, et al., Hum Genet, 96:275-80, 1995) or comparative genomichybridization (CGH) (Forozan, et al., Trends Genet, 13:405-9, 1997;Thompson and Gray, J Cell Biochem Suppl, 13943, 1993; Houldsworth andChaganti, Am J Pathol, 145:1253-60, 1994); (d) isothermic DNAamplification (Lizardi et al., Nat Genet, 19:225-32, 1998; Mitra andChurch, Nucleic Acids Res, 27:e34, 1999); and (e) quantitativehybridization to an immobilized probe(s) (Southern, J. Mol. Biol. 98:503, 1975; Kafatos et al., Nucleic Acids Res 7(6):1541-52, 1979).

Applicants provide herein an example in which TAQMAN® quantitative PCRis used to screen for successfully targeted eukaryotic cells. Forexample, TAQMAN® is used to identify eukaryotic cells which haveundergone homologous recombination wherein a portion of one of twoendogenous alleles in a diploid genome has been replaced by anothersequence. In contrast to traditional methods, in which a difference inrestriction fragment length spanning the entire homology arm or armsindicates the modification of one of two alleles, the quantitativeTAQMAN® method will detect the modification of one allele by measuringthe reduction in copy number (by half) of the unmodified allele.Specifically, the probe detects the unmodified allele and not themodified allele. Therefore, the method is independent of the exactnature of the modification and not limited to the sequence replacementdescribed in this example. TAQMAN® is used to quantify the number ofcopies of a DNA template in a genomic DNA sample, especially bycomparison to a reference gene (Lie and Petropoulos, Curr. Opin.Biotechnol., 9:43-8, 1998). The reference gene is quantitated in thesame genomic DNA as the target gene(s) or locus (loci). Therefore, twoTAQMAN® amplifications (each with its respective probe) are performed.One TAQMAN® probe determines the “Ct” (Threshold Cycle) of the referencegene, while the other probe determines the Ct of the region of thetargeted gene(s) or locus (loci) which is replaced by successfultargeting. The Ct is a quantity that reflects the amount of starting DNAfor each of the TAQMAN® probes, i.e. a less abundant sequence requiresmore cycles of PCR to reach the threshold cycle. Decreasing by half thenumber of copies of the template sequence for a TAQMAN® reaction willresult in an increase of about one Ct unit. TAQMAN® reactions in cellswhere one allele of the target gene(s) or locus (loci) has been replacedby homologous recombination will result in an increase of one Ct for thetarget TAQMAN® reaction without an increase in the Ct for the referencegene when compared to DNA from non-targeted cells. This allows for readydetection of the modification of one allele of the gene(s) of interestin eukaryotic cells using LTVECs.

As stated above, modification of allele (MOA) screening is the use ofany method that detects the modification of one allele to identify cellswhich have undergone homologous recombination. It is not a requirementthat the targeted alleles be identical (homologous) to each other, andin fact, they may contain polymorphisms, as is the case in progenyresulting from crossing two different strains of mice. In addition, onespecial situation that is also covered by MOA screening is targeting ofgenes which are normally present as a single copy in cells, such as someof the located on the sex chromosomes and in particular, on the Ychromosome. In this case, methods that will detect the modification ofthe single targeted allele, such as quantitative PCR, Southernblottings, etc., can be used to detect the targeting event. It is clearthat the method of the invention can be used to generate modifiedeukaryotic cells even when alleles are polymorphic or when they arepresent in a single copy in the targeted cells.

Step 8. Uses of Genetically Modified Eukaryotic Cells. (a) Thegenetically modified eukaryotic cells generated by the methods describedin steps 1 through 7 can be employed in any in vitro or in vivo assay,where changing the phenotype of the cell is desirable. (b) Thegenetically modified eukaryotic cell generated by the methods describedin steps 1 through 7 can also be used to generate an organism carryingthe genetic modification. The genetically modified organisms can begenerated by several different techniques including but not limitedto: 1. Modified embryonic stem (ES) cells such as the frequently usedrat and mouse ES cells. ES cells can be used to create geneticallymodified rats or mice by standard blastocyst injection technology oraggregation techniques (Robertson, Practical Approach Series, 254, 1987;Wood, et al., Nature, 365:87-9, 1993; Joyner supra), tetraploidblastocyst injection (Wang, et al., Mech Dev, 62:137-45, 1997), ornuclear transfer and cloning (Wakayama, et al., Proc Natl Acad Sci USA,96:14984-9, 1999). ES cells derived from other organisms such as rabbits(Wang, et al., Mech Dev, 62:137-45, 1997; Schoonjans, et al., Mol ReprodDev, 45:439-43, 1996) or chickens (Pain, et al., Development,122:2339-48, 1996) or other species should also be amenable to geneticmodification(s) using the methods of the invention. 2. Modifiedprotoplasts can be used to generate genetically modified plants (forexample see U.S. Pat. No. 5,350,689 “Zea mays plants and transgenic Zeamays plants regenerated from protoplasts or protoplast-derived cells”,and U.S. Pat. No. 5,508,189 “Regeneration of plants from cultured guardcell protoplasts” and references therein). 3. Nuclear transfer frommodified eukaryotic cells to oocytes to generate cloned organisms withmodified allele (Wakayama, et al., Proc Natl Acad Sci USA, 96:14984-9,1999; Baguisi, et al., Nat Biotechnol, 17:456-61, 1999; Wilmut, et al.,Reprod Fertil Dev, 10:639-43, 1998; Wilmut, et al., Nature, 385:810-3,1997; Wakayama, et al., Nat Genet, 24:108-9, 2000; Wakayama, et al.,Nature, 394:369-74, 1998; Rideout, et al., Nat Genet, 24:109-10, 2000;Campbell, et al., Nature, 380:64-6, 1996). 4. Cell-fusion to transferthe modified allele to another cell, including transfer of engineeredchromosome(s), and uses of such cell(s) to generate organisms carryingthe modified allele or engineered chromosome(s) (Kuroiwa, et al., NatBiotechnol, 18:1086-1090, 2000). 5. The method of the invention are alsoamenable to any other approaches that have been used or yet to bediscovered.

While many of the techniques used in practicing the individual steps ofthe methods of the invention are familiar to the skilled artisan,Applicants contend that the novelty of the method of the invention liesin the unique combination of those steps and techniques coupled with thenever-before-described method of introducing a LTVEC directly intoeukaryotic cells to modify a chromosomal locus, and the use ofquantitative MOA assays to identify eukaryotic cells which have beenappropriately modified. This novel combination represents a significantimprovement over previous technologies for creating organisms possessingmodifications of endogenous genes or chromosomal loci.

EXAMPLES Example 1

Engineering Mouse ES Cells Bearing a Deletion of the OCR10 Gene

a. Selection of a Large Genomic DNA Clone Containing mOCR10. A BacterialArtificial Chromosome (BAC) clone carrying a large genomic DNA fragmentthat contained the coding sequence of the mouse OCR10 (mOCR10) gene wasobtained by screening an arrayed mouse genomic DNA BAC library (IncyteGenomics) using PCR. The primers employed to screen this library werederived from the mOCR10 gene cDNA sequence. Two primer pairs where used:(a) OCR10.RAA (SEQ ID NO:1) and OCR10.PVIrc (SEQ ID NO:2) whichamplifies a 102 bp DNA; and (b) OCR10.TDY (SEQ ID NO:3)) and OCR10.QETrc(SEQ ID NO:4)) which amplifies a 1500 bp DNA. This mOCR10 BAC containedapproximately 180 kb of genomic DNA including the complete mOCR10 codingsequence. This BAC clone was used to generate an LTVEC which wassubsequently used to delete a portion of the coding region of mOCR10while simultaneously introducing a reporter gene whose initiation codonprecisely replaced the initiation codon of OCR10, as well as insertionof a selectable marker gene useful for selection both in E. coli andmammalian cells following the reporter gene (FIG. 2). The reporter gene(LacZ), encodes the E. coli β-galactosidase enzyme. Because of theposition of insertion of LacZ (its initiating codon is at the sameposition as the initiation codon of mOCR10) the expression of lacZshould mimic that of mOCR10, as has been observed in other exampleswhere similar replacements with LacZ were performed using previoustechnologies (see “Gene trap strategies in ES cells”, by W Wurst and A.Gossler, in Joyner supra). The LacZ gene allows for a simple andstandard enzymatic assay to be performed that can reveal its expressionpatterns in situ, thus providing a surrogate assay that reflects thenormal expression patterns of the replaced gene(s) or chromosomal locus(loci).

b. Construction of Donor Fragment and Generation of LTVEC. Themodification cassette used in the construction of the mOCR10 LTVEC isthe lacZ-SV40 polyA-PGKp-EM7-neo-PGK polyA cassette wherein lacZ is amarker gene as described above, SV40 polyA is a fragment derived fromSimian Virus 40 (Subramanian, et al., Prog Nucleic Acid Res Mol Biol,19:157-64, 1976; Thimmappaya, et al., J Biol Chem, 253:1613-8, 1978;Dhar, et al., Proc Natl Acad Sci USA, 71:371-5, 1974; Reddy, et al.,Science, 200:494-502, 1978) and containing a polyadenylation site andsignal (Subramanian, et al., Prog Nucleic Acid Res Mol Biol, 19:157-64,1976; Thimmappaya, et al., J Biol Chem, 253:1613-8, 1978; Dhar, et al.,Proc Natl Acad Sci USA, 71:371-5, 1974; Reddy, et al., Science,200:494-502, 1978), PGKp is the mouse phosphoglycerate kinase (PGK)promoter (Adra, et al., Gene, 60:65-74, 1987) (which has been usedextensively to drive expression of drug resistance genes in mammaliancells), EM7 is a strong bacterial promoter that has the advantage ofallowing for positive selection in bacteria of the completed LTVECconstruct by driving expression of the neomycin phosphotransferase (neo)gene, neo is a selectable marker that confers Kanamycin resistance inprokaryotic cells and G418 resistance in eukaryotic cells (Beck, et al.,Gene, 19:327-36, 1982), and PGK polyA is a 3′ untranslated regionderived from the PGK gene and containing a polyadenylation site andsignal (Boer, et al., Biochem Genet, 28:299-308, 1990).

To construct the mOCR10 LTVEC, first a donor fragment was generatedconsisting of a mOCR10 homology box 1 (hb1) attached upstream from theLacZ gene in the modification cassette and a mOCR10 homology box 2 (hb2)attached downstream of the neo-PGK polyA sequence in the modificationcassette (FIG. 2), using standard recombinant genetic engineeringtechnology. Homology box 1 (hb1) consists of 211 bp of untranslatedsequence immediately upstream of the initiating methionine of the mOCR10open reading frame (mOCR10 ORF) (FIG. 3A-3D). Homology box 2 (hb2)consists of last 216 bp of the mOCR10 ORF, ending at the stop codon(FIG. 3A-3D).

Subsequently, using bacterial homologous recombination (Zhang, et al.supra; Angrand, et al., supra; Muyrers, et al. supra; Narayanan et al.supra; Yu et al. supra), this donor fragment was used to preciselyreplace the mOCR10 coding region (from initiation methionine to stopcodon) with the insertion cassette, resulting in construction of themOCR10 LTVEC (FIG. 2). Thus, in this mOCR10 LTVEC, the mOCR10 codingsequence was replaced by the insertion cassette creating anapproximately 20 kb deletion in the mOCR10 locus while leavingapproximately 130 kb of upstream homology (upstream homology arm) and 32kb of downstream homology (downstream homology arm).

It is important to note that LTVECs can be more rapidly and convenientlygenerated from available BAC libraries than targeting vectors made usingprevious technologies because only a single bacterial homologousrecombination step is required and the only sequence informationrequired is that needed to generate the homology boxes. In contrast,previous approaches for generating targeting vectors using bacterialhomologous recombination require that large targeting vectors be“trimmed” prior to their introduction in ES cells (Hill et al.,Genomics, 64:111-3, 2000). This trimming is necessary because of theneed to generate homology arms short enough to accommodate the screeningmethods utilized by previous approaches. One major disadvantage of themethod of Hill et al. is that two additional homologous recombinationsteps are required simply for trimming (one to trim the region upstreamof the modified locus and one to trim the region downstream of themodified locus). To do this, substantially more sequence information isneeded, including sequence information spanning the sites of trimming.

In addition, another obvious advantage, illustrated by the aboveexample, is that a very large deletion spanning the mOCR10 gene(approximately 20 kb) can be easily generated in a single step. Incontrast, using previous technologies, to accomplish the same task mayrequire several steps and may involve marking the regions upstream anddownstream of the coding sequences with loxP sites in order to use theCre recombinase to remove the sequence flanked by these sites afterintroduction of the modified locus in eukaryotic cells. This may beunattainable in one step, and thus may require the construction of twotargeting vectors using different selection markers and two sequentialtargeting events in ES cells, one to introduce the loxP site at theregion upstream of the coding sequence and another to introduce the loxPsite at the region downstream of the coding sequence. It should befurther noted that the creation of large deletions often occurs with lowefficiency using the previous targeting technologies in eukaryoticcells, because the frequency of achieving homologous recombination maybe low when using targeting vectors containing large deletion flanked byrelatively short homology arms. The high efficiency obtained using themethod of the invention (see below) is due to the very long homologyarms present in the LTVEC that increase the rate of homologousrecombination in eukaryotic cells.

c. Verification, Preparation, and Introduction of mOCR10 LTVEC DNA intoES Cells. The sequence surrounding the junction of the insertioncassette and the homology sequence was verified by DNA sequencing. Thesize of the mOCR10 LTVEC was verified by restriction analysis followedby pulsed field gel electrophoresis (PFGE) (Cantor, et al., Annu RevBiophys Biophys Chem, 17:287-304, 1988; Schwartz and Cantor, Cell,37:67-75, 1984). A standard large-scale plasmid preparation of themOCR10 LTVEC was done, the plasmid DNA was digested with the restrictionenzyme NotI, which cuts in the vector backbone of the mOCR10 LTVEC, togenerate linear DNA. Subsequently the linearized DNA was introduced intomouse ES cells by electroporation (Robertson, Practical Approach Series,254, 1987; Joyner supra; Sambrook, et al. supra). ES cells successfullytransfected with the mOCR10 LTVEC were selected for in G418-containingmedia using standard selection methods.

d. Identification of Targeted ES Cells Clones Using a QuantitativeModification of Allele (MOA) Assay. To identify ES cells in which one ofthe two endogenous mOCR10 genes had been replaced by the modificationcassette sequence, DNA from individual ES cell clones was analyzed byquantitative PCR using standard TAQMAN® methodology as described(Applied Biosystems, TAQMAN® Universal PCR Master Mix, catalog numberP/N 4304437). The primers and TAQMAN® probes used are as described inFIG. 3A-3D (SEQ ID NO:5-6). A total of 69 independent ES cells cloneswhere screened and 3 were identified as positive, i.e. as clones inwhich one of the endogenous mOCR10 coding sequence had been replaced bythe modification cassette described above.

Several advantages of the MOA approach are apparent: (i) It does notrequire the use of a probe outside the locus being modified, thusobviating the need to know the sequence flanking the modified locus.(ii) It requires very little time to perform compared to conventionalSouthern blot methodology which has been the previous method of choice,thus reducing the time for identifying correctly modified cells from thetypical several days to just a few hours. This is a significantimprovement in the way screening has been performed in the past andmakes it a much less labor-intensive and more cost-effective approach toscreening for homologous recombination events in eukaryotic cells. Yetanother advantage of the method of the invention is that it is alsosuperior to previous technologies because of its ability to targetdifficult loci. Using previous technologies, it has been shown that forcertain loci the frequency of successful targeting may by as low as 1 in2000 integration events, perhaps even lower. Using the method of theinvention, Applicants have demonstrated that such difficult loci can betargeted much more efficiently using LTVECs that contain long homologyarms (i.e. greater than those allowed by previous technologies). As thenon-limiting example described above demonstrates, the Applicants havetargeted the OCR10 locus, a locus that has previously provenrecalcitrant to targeting using conventional technology. Using themethod of the invention, Applicants have shown that they have obtainedsuccessful targeting in 3 out of 69 ES cells clones in which the mOCR10LTVEC (containing more than 160 kb of homology arms, and introducing a20 kb deletion) had integrated, whereas using previous technology for EScell targeting using a plasmid-based vector with homology arms shorterthan 10-20 kb while also introducing a deletion of less than 15 kb, notargeted events were identified among more than 600 integrants of thevector. These data clearly demonstrate the superiority of the method ofthe invention over previous technologies.

Example 2

Increased Targeting Frequency and Abrogation of the Need to Use IsogenicDNA when LTVECs are Used as the Targeting Vectors

As noted above, the increased targeting frequency obtained using longhomology arms should diminish the benefit, if any, derived from usinggenomic DNA in constructing LTVECs that is isogenic with (i.e. identicalin sequence to) the DNA of the eukaryotic cell being targeted. To testthis hypothesis, Applicants have constructed numerous LTVECs usinggenomic DNA derived from the same mouse substrain as the eukaryotic cellto be targeted (presumably isogenic), and numerous other LTVECs usinggenomic DNA derived from mouse substrains differing from that of theeukaryotic cell to be targeted (presumably non-isogenic). The two setsof LTVECs exhibited similar targeting frequencies, ranging from 1-13%,indicating that the rate of successful targeting using LTVECs does notdepend on isogenicity.

The approach of creating LTVECs and directly using them as targetingvectors combined with MOA screening for homologous recombination eventsin ES cells creates a novel method for engineering genetically modifiedloci that is rapid, inexpensive and represents a significant improvementover the tedious, time-consuming methods previously in use. It thusopens the possibility of a rapid large scale in vivo functional genomicsanalysis of essentially any and all genes in an organism's genome in afraction of the time and cost necessitated by previous methodologies.

Example 3

Use of LTVECs to Produce Chimeric and Human Antibodies

a. Introduction. The rearrangement of variable region genes during theinitial development of B cells is the primary mechanism whereby theimmune system produces antibodies capable of recognizing the huge numberof antigens that it may encounter. Essentially, through DNArearrangements during B cell development, a huge repertoire of variable(V) region sequences are assembled which are subsequently joined to aconstant (C) region to produce complete heavy and light chains whichassemble to form an antibody. After functional antibodies have beenassembled, somatic hypermutation which occurs in the secondary lymphoidorgans, introduces further diversity which enables the organism toselect and optimize the affinity of the antibody.

The production of antibodies to various antigens in non-human speciesinitially provided great promise for the large scale production ofantibodies that could be used as human therapeutics. Speciesdifferences, however, leads to the production of antibodies by humanswhich inactivate the foreign antibodies and cause allergic reactions.Attempts were subsequently made to “humanize” the antibodies, thusmaking them less likely to be recognized as foreign in humans.Initially, this process involved combining the antigen binding portionsof antibodies derived from mice with the constant region of humanantibodies, thereby creating recombinant antibodies that were lessimmunogenic in humans. A second approach which was developed was phagedisplay, whereby human V regions are cloned into a phage display libraryand regions with the appropriate binding characteristics are joined tohuman constant regions to create human antibodies. This technology islimited, however, by the lack of antibody development and affinitymaturation which naturally occurs in B cells.

More recently, endogenous genes have been knocked out of mice, and thegenes replaced with their human counterparts to produce entirely humanantibodies. Unfortunately, the use of these constructs has highlightedthe importance of an endogenous constant region in the development andoptimization of antibodies in B cells. Human antibodies produced bytransgenic mice with entirely human constructs have reduced affinity ascompared to their mouse counterparts. Accordingly, the much acclaimedmethods of producing humanized antibodies in mice and other organisms,wherein endogenous variable and constant regions of the mice are knockedout and replaced with their human counterparts, has not resulted inoptimal antibodies.

The use of chimeric antibodies, which utilize human variable regionswith mouse constant regions through B cell maturation, followed bysubsequent engineering of the antibodies to replace the mouse constantregions with their human counterparts, has been suggested (U.S. Pat. No.5,770,429). However, the only methodology that has existed to date formaking such chimeras has been trans-switching, wherein the formation ofthe chimeras is only a rare event which occurs only in heavy chains.Heretofore, there has been no mechanism to produce, in transgenicanimals, large scale replacement of the entire variable gene encodingsegments with human genes, thereby producing chimeras in both the heavyand light chains. Utilizing Applicants' technology, as disclosed herein,chimeric antibodies are generated which can then be altered, throughstandard technology, to create high affinity human antibodies.

b. Brief Description. A transgenic mouse is created that produces hybridantibodies containing human variable regions and mouse constant regions.This is accomplished by a direct, in situ replacement of the mousevariable region genes with their human counterparts. The resultanthybrid immunoglobulin loci will undergo the natural process ofrearrangements during B-cell development to produce the hybridantibodies.

Subsequently, fully-human antibodies are made by replacing the mouseconstant regions with the desired human counterparts. This approach willgive rise to therapeutic antibodies much more efficiently than previousmethods, e.g. the “humanization” of mouse monoclonal antibodies or thegeneration of fully human antibodies in HUMAB™ mice. Further, thismethod will succeed in producing therapeutic antibodies for manyantigens for which previous methods have failed. This mouse will createantibodies that are human variable region-mouse constant region, whichwill have the following benefits over the previously available HUMAB™mice that produce totally human antibodies. Antibodies generated by thenew mouse will retain murine Fc regions which will interact moreefficiently with the other components of the mouse B cell receptorcomplex, including the signaling components required for appropriate Bcell differentiation (such as Iga and Igb). Additionally, the murine Fcregions will be more specific than human Fc regions in theirinteractions with Fc receptors on mouse cells, complement molecules,etc. These interactions are important for a strong and specific immuneresponse, for the proliferation and maturation of B cells, and for theaffinity maturation of antibodies.

Because there is a direct substitution of the human V-D-J/V-J regionsfor the equivalent regions of the mouse loci all of the sequencesnecessary for proper transcription, recombination, and/or classswitching will remain intact. For example, the murine immunoglobulinheavy chain intronic enhancer, Em, has been shown to be critical forV-D-J recombination as well as heavy chain gene expression during theearly stages of B cell development (Ronai et al. Mol Cell Biol19:7031-7040 (1999)), whereas the immunoglobulin heavy chain 3′ enhancerregion appears to be critical for class switching (Pan et al. Eur JImmunol 30:1019-1029 (2000)) as well as heavy chain gene expression atlater stages of B cell differentiation (Ong, et al. J Immunol160:4896-4903 (1998)). Given these various, yet crucial, functions ofthe transcriptional control elements, it is desirable to maintain thesesequences intact.

The required recombination events which occur at the immunoglobulin lociduring the normal course of B cell differentiation may increase thefrequency of aberrant, non-productive immunoglobulin rearrangements whenthese loci are inserted at improper chromosomal locations, or inmultiple copies, as in currently available mice. With reductions inproductive immunoglobulin rearrangement and, therefore, appropriatesignaling at specific steps of B cell development the aberrant cells areeliminated. Reductions of B cell numbers at early stages of developmentsignificantly decreases the final overall B cell population and greatlylimits the immune responses of the mice. Since there will be only one,chimeric, heavy or light chain locus (as opposed to mutatedimmunoglobulin loci and with human transgenic loci integrated atdistinct chromosomal locations for heavy and light chains in thecurrently available mice) there should be no trans-splicing ortrans-rearrangements of the loci which could result in non-productiverearrangements or therapeutically irrelevant chimeric antibodies(Willers et al. Immunobiology 200:150-164 (2000); Fujieda et al. J.Immunol. 157:3450-3459 (1996)).

The substitutions of the human V-D-J or V-J regions into the genuinemurine chromosomal immunoglobulin loci should be substantially morestable, with increased transmission rates to progeny and decreasedmosaicism of B cell genotypes compared with the currently available mice(Tomizuka et al Proc Natl Acad Sci (USA) 97:722-727 (2000)).Furthermore, introduction of the human variable regions at the genuinemurine loci in vivo will maintain the appropriate global regulation ofchromatin accessibility previously shown to be important forappropriately timed recombination events (Haines et al. Eur J Immunol28:4228-4235 (1998)).

Approximately ⅓ of human antibodies contain lambda light chains, ascompared to mice in which only 1/20 of murine antibodies contain lambdalight chains. Therefore, replacing murine lambda light chain V-Jsequences with lambda light chain V-J sequences derived from the humanlocus will serve to increase the repertoire of antibodies as well asmore closely match the genuine human immune response, thus increasingthe likelihood of obtaining therapeutically useful antibodies.

An additional benefit of integrating the human sequences into thegenuine murine immunoglobulin loci is that no novel integration sitesare introduced which might give rise to mutagenic disruptions at theinsertion site and preclude the isolation of viable homozygous mice.This will greatly simplify the production and maintenance of a breedingmouse colony.

c. Materials and Methods: Precise replacement of the mouse heavy chainlocus variable region (VDJ) with its human counterpart is exemplifiedusing a combination of homologous and site-specific recombination in thefollowing example, which utilizes a two step process. One skilled in theart will recognize that replacement of the mouse locus with thehomologous or orthologous human locus may be accomplished in one or moresteps. Accordingly, the invention contemplates replacement of the murinelocus, in whole or in part, with each integration via homologousrecombination.

Large insert (BAC) clones spanning the entire VDJ region of the humanheavy chain locus are isolated (FIG. 4A). The sequence of this entireregion is available in the following Gen Bank files (AB019437, AB019438,AB019439, AB019440, AB019441, X97051 and X54713). In this example, largeinsert (BAC) clones are isolated from the ends of the mouse VDJ regionas a source of homology arms (FIG. 4B) which are used to directintegration via homologous recombination of the human VDJ sequences in atwo step process.

In the first step, LTVEC1 (FIG. 4D) is constructed by bacterialhomologous recombination in E. coli. LTVEC1 contains, in order: a largemouse homology arm derived from the region upstream from the mouse DJregion, but whose absolute endpoints are not important; a cassetteencoding a selectable marker functional in ES cells (PGK-neomycinR); aloxP site; a large human insert spanning from several V gene segmentsthrough the entire DJ region; and a mouse homology arm containing theregion immediately adjacent to, but not including, the mouse J segments.Mouse ES cells will be transformed by standard techniques, for example,electroporation, with linearized LTVEC1, and neomycin resistant colonieswill be screened for correct targeting using a MOA assay. These targetedES cells can give rise to mice that produce antibodies with hybrid heavychains. However, it will be preferable to proceed with subsequent stepsthat will eliminate the remainder of the mouse variable segments.

In the second step, LTVEC2 (FIG. 4C) is constructed by bacterialhomologous recombination in E. coli. LTVEC2 contains, in order: a largemouse homology arm containing the region adjacent to the most distalmouse V gene segment, but not containing any mouse V gene segments; alarge insert containing a large number of distal human V gene segments;a mutant loxP site called lox511 (Hoess et al. Nucleic Acids Res.14:2287-2300 (1986)), in the orientation opposite to that of the wildtype loxP sites in LTVEC2 and LTVEC1 (this site will not recombine withwild type loxP sites but will readily recombine with other lox511sites); a wild type loxP site; a second selectable marker(PGK-hygromycinR); and a mouse homology arm derived from the V region,but whose absolute endpoints are not important. Mouse ES cells that werecorrectly targeted with LTVEC1 will then be transformed by standardtechniques with linearized LTVEC2, and hygromycin resistant colonieswill be screened for correct targeting using a MOA assay. Correctlytargeted ES cells resulting from this transformation will hereafter bereferred to as “double targeted ES cells”.

Subsequent transient expression of CRE recombinase in the doubletargeted ES cells will result in deletion of the remainder of the mouseV region. Alternatively, the double targeted ES cells can be injectedinto host blastocysts for the production of chimeric mice. Breeding ofthe resultant chimeric mice with mice expressing CRE recombinase earlyin development will result in deletion of the remainder of the mouse Vregion in the progeny F1. This later alternative increases thelikelihood that the hybrid heavy chain locus will be passed through thegermline because it involves culturing the ES cells for fewergenerations.

The inclusion of lox511 in LTVEC2 will allow for the insertion ofadditional human V gene segments into the hybrid locus. One approachwould be to use bacterial homologous recombination to flank a largegenomic DNA clone containing many additional human V gene segments withlox511 and loxP sites. Co-transformation of such a modified largegenomic DNA clone into double targeted ES cells with a plasmid thattransiently expresses CRE recombinase will result in the introduction ofthe additional V gene segments by cassette exchange (Bethke et al.Nucleic Acids Res. 25:2828-2834 (1997)).

A second approach to the incorporation of additional V gene segments isto independently target a large genomic DNA clone containing manyadditional human V gene segments into the mouse locus using, forinstance, the same mouse homology arms included in LTVEC2. In this case,the additional human V gene segments would be flanked by lox511 and loxPsites, and the targeted ES cells would be used to create a mouse. Themice derived from double targeted ES cells and the mice derived from theES cells containing the additional V gene segments would be bred with athird mouse that directs expression of CRE recombinase during meiosis.The close proximity of the two recombinant loci during meiotic pairingwould result in a high frequency of CRE induced inter-chromosomalrecombination as has been seen in other systems (Herault et al. NatureGenetics 20: 381-384 (1998)).

The final steps in creating the human variable/mouse constant monoclonalantibody producing-mouse will be performing the equivalent variableregion substitutions on the lambda and kappa light chain loci andbreeding all three hybrid loci to homozygocity together in the samemouse. The resultant transgenic mouse will have a genome comprisingentirely human heavy and light chain variable gene loci operably linkedto entirely endogenous mouse constant region such that the mouseproduces a serum containing an antibody comprising a human variableregion and a mouse constant region in response to antigenic stimulation.Such a mouse may then be used as a source of DNA encoding the variableregions of human antibodies. Using standard recombinant technology, DNAencoding the variable regions of the heavy and light chains of theantibody is operably linked to DNA encoding the human heavy and lightchain constant regions in cells, such as a CHO cells, which are capableof expressing active antibodies. The cells are grown under theappropriate conditions to express the fully human antibodies, which arethen recovered. Variable region encoding sequences may be isolated, forexample, by PCR amplification or cDNA cloning. In a preferredembodiment, hybridomas made from transgenic mice comprising some or allof the human variable region immunoglobulin loci (Kohler et al. Eur. J.Immunol., 6:511-519 (1976) are used as a source of DNA encoding thehuman variable regions.

We claim:
 1. A transgenic mouse whose genome comprises unrearrangedhuman immunoglobulin heavy chain V, D, and J gene segments contained ona human genomic DNA fragment that is greater than 100 kb, wherein theunrearranged human immunoglobulin heavy chain V, D, and J gene segmentsin situ replace endogenous mouse immunoglobulin heavy chain V, D, and Jgene segments, and the unrearranged human immunoglobulin heavy chain V,D, and J gene segments are operably linked to an endogenous mouse heavychain constant region gene, wherein the mouse heavy chain constantregion gene is located at an endogenous mouse immunoglobulin heavy chainconstant region locus, wherein the unrearranged human immunoglobulinheavy chain V, D, and J gene segments are present in the germline of themouse, wherein rearrangement of the human immunoglobulin heavy chain V,D, and J gene segments in the mouse results in a rearranged human heavychain variable region gene operably linked to the mouse heavy chainconstant region gene, wherein the mouse in response to an antigenproduces a hybrid antibody that comprises a human heavy chain variableregion encoded by the rearranged human heavy chain variable region geneand a murine immunoglobulin Fc region encoded by the mouse heavy chainconstant region gene, wherein mouse V gene segments are present upstreamof the unrearranged human heavy chain immunoglobulin V, D, and J genesegments, and wherein the endogenous immunoglobulin enhancer Eμ remainsintact upstream of the endogenous mouse heavy chain immunoglobulinconstant region gene.
 2. The mouse of claim 1, wherein the mouse doesnot produce fully human antibodies.